Conventionally, in order to detect a biomolecule such as nucleic acid, protein, peptide, enzyme, receptor, antibody, sugar, fat, vitamin, hormone, or a substance related thereto, the biomolecule is immobilized onto an appropriate plate, and antigen, fluorescent material, luminescent material, or the like having high affinity with the immobilized biomolecule is dropped onto the biomolecule, and thereafter, the biomolecule immobilized onto the plate is detected by an optical detection method such as absorptiometry, fluorometry, or emission spectrometry utilizing antigen-antibody reaction, fluorogenic reaction, chemiluminascence reaction, or electrochemical reaction. Since the method of detecting biomolecules immobilized on a plate significantly facilitates addition and removal of reagents during detection of biomolecules, various kinds of immobilization plates have been proposed and put to practical use.
For example, there have been proposed, as immobilization plates, a nitrocellulose film and a nylon film which make hydrophobic bonding to nucleic acid, protein, or the like. To this day, detection of nucleic acid by the southern blotting method or northern blotting method and detection of protein by the western blotting method have been carried out using such nitrocellulose film or nylon film as an immobilization plate.
Further, a plastic plate (micro plate) has also been used as an immobilization plate. For example, a micro plate of about 80×120 mm having 96 pieces of dents, each dent having a diameter of about 7 mm and a depth of about 10 mm, is used, and the surface of the plate with the dents is made to have hydrophobicity, or functional groups are introduced onto the surface, whereby nucleic acid or protein is immobilized by hydrophobic bonding or ion bonding. Since this micro plate enables simultaneous processing of plural specimens, it is utilized for biomolecule detection using the enzyme immunoassay (EIA) or the fluorescent immunoassay (FIA).
Further, the method of bonding biomolecules onto a plate by hydrophobic boding or ion bonding is also applied to microarray technique which has recently been contrived. The microarray technique is a technique of immobilizing 1000 or more biomolecules on a plate such as a slide glass having a size of about 25 mm×75 mm. For example, a DNA probe is immobilized on a slide glass, and the immobilized DNA probe and a labeled target DNA are subjected to hybridization, whereby the target DNA can be speedily detected with high sensitivity (refer to, for example, Japanese Patent No. 3272365; Fodor, S. P. A. et al., Science, 1991, Vol. 251, P. 767; Schena, M et al., Science, 1995, Vol. 270, P. 467). In the micro array technique, glass is mainly used as a material of a plate and, at the beginning, immobilization utilizing ion bonding with biomolecules was employed. However, in the immobilization method utilizing hydrophobic bonding and ion bonding, the immobilized biomolecules might be dissociated from the plate during the process steps of addition of a reaction reagent, washing, and emission, resulting in degradation of bonding stability. Therefore, there is proposed a method in which functional groups are produced on the surface of the glass plate, and the glass plate and the biomolecules are covalently bonded through the functional groups.
For example, there is proposed a method in which, after the surface of a glass plate is oxidized and hydrophilic groups are introduced, an aminosilane coupling agent is condensation-reacted with the hydrophilic groups, thereby to introduce amino groups as functional groups onto the surface of the glass plate (for example, refer to Japanese Patent No. 1649351).
Further, there is proposed a method of introducing carboxyl groups as functional groups onto the surface of a glass plate, using a silane compound that is highly reactive to hydroxyl groups on the surface of the glass plate (for example, Japanese Published Patent Application No. 2001-108683). Since the silane compound is a material having a high degree of reactivity to glass, it is frequently used for introduction of functional groups onto the surface of the glass plate, and further, it is known that the variation in smoothness of the glass plate can be maintained within about 3 nm even after the silane compound processing (for example, refer to Telechem International Inc. website at http://www.arrayit.com/Products/Substrates/SMA/sma.tml, which is searched on December, 2003).
Further, there are also proposed a method of introducing functional groups by coating the surface of a plate with a polymer (for example, refer to Japanese published Patent Application No. 2001-518604), and a method of introducing carbide as functional groups by vapor-depositing diamond like carbon on the surface of a glass plate (for example, refer to Japanese Published Patent Applications No. 2002-82116 and No. 2002-350440).
Moreover, there is proposed a method of immobilizing gene as biomolecule on the surface of a plate using a microarray technique for gene detection. For example, there is proposed a method in which, after nucleotide is immobilized on the surface of a silicon substrate, the nucleotide having light-polymerized functional groups is successively extended by an application of a semiconductor processing technique, thereby to produce oligonucleotide (for example, refer to Japanese Published Patent Application No. 2000-508542, or Lipshutz, R. J. et al., Nature Genetics supplement, 1999, Vol. 21, P. 20). Further, there is proposed a method in which nucleotide is dropped on a glass plate and then the nucleotide is successively extended by an application of an ink-jet printer technique (for example, refer to Japanese Published Patent Application No. 2001-50960).
In immobilization of biomolecules on a plate, uniformity of the amount of immobilized biomolecules per unit area of the plate is required. In addition, since the immobilized biomolecules are detected utilizing the optical method such as the absorptiometry, fluorometry, or emission spectrometry, it is required that the optical characteristics of the plate itself, such as optical transparency, light scattering, light absorption, and light refraction, should be uniform. Further, it is also required that the smoothness of the surface of the plate itself should be uniform. However, the conventional technique has the following drawbacks with respect to these requirements.
Initially, when a microplate (plastic plate) is used as an immobilization plate, the optical characteristics and smoothness of the surface having dents, onto which biomolecules are immobilized must be uniform. In this case, however, the surface having dents must be subjected to an appropriate physical or chemical processing to make the surface have immobilization ability, and therefore, it is difficult to uniformly maintain the optical characteristics and smoothness of the plate surface until finishing the immobilization process.
On the other hand, although there have been proposed the method of processing a plate with silane compound, the method of coating the surface of a plate with polymer, and the method of vapor-depositing diamond like carbon onto the surface of glass as methods for producing functional groups on the plates to firmly immobilize biomolecules on the plates such as glass, these methods also have drawbacks as follows. Since the silane compound has a high deactivity, introduction of the functional groups is not stably carried out, and thereby the amount of immobilized biomolecules is not uniform. Further, when polymer is coated over the plate surface, unevenness occurs due to the coating layer, and thereby the optical characteristics, the smoothness, and the number of functional groups per unit area (the amount of immobilized biomolecules) of the plate surface are not uniform. Further, when diamond like carbon is vapor-deposited on the surface of the glass plate, the deposited carbon forms a polycrystalline structure, and thereby the plate surface is difficult to be smooth.
Further, in the method of immobilizing biomolecules by introducing functional groups onto glass plates or the like as described above, some functional groups that do not react with desired biomolecules may be adsorbed to or combined with undesired biomolecules, and this may cause noise components during measurement.
Furthermore, the method of immobilizing gene on a silicon substrate by utilizing the semiconductor processing technique enables highly accurate immobilization of gene. However, since this method utilizes expanding formation of nucleotide on the substrate, it is only applicable to gene which comprises a combination of four kinds of bases, and it is difficult to apply this method to immobilization of other biomolecules. Further, the above-mentioned method of immobilizing gene using the ink-jet technique also has similar problems.
Furthermore, in the microarray technique of immobilizing 1000 or more specimens on a glass-slide-sized glass plate, since there are great number of specimens per unit area which are to be immobilized on the biomolecule-immobilized plate, the diameter of a spot corresponding to one specimen becomes several tens of microns, and thereby the amount of immobilized biomolecules per specimen becomes very small. Therefore, when using the microarray technique, in order to improve the detection accuracy, not only uniformity of the optical characteristics of the plate itself but also formation of a highly smooth plate having no positional irregularity, no strains, and no minute flaws are required. Accordingly, a very expensive plate as compared with a slide glass plate used for microscopic observation or the like must be used. For example, when silicon is used as a plate, high smoothness can be obtained as is evident from the semiconductor fabrication method. However, a silicon plate is expensive. So, an inexpensively producible plate alternative to glass or silicon has been desired. Moreover, in the microarray technique, when biomolecules are immobilized on a plate such as a glass, a liquid droplet containing the biomolecules is applied by a pin spotter or the like. So, if functional groups are previously introduced over the surface of the plate, the functional groups may cause positional deviation and uneven shape of the spot, resulting in a reduction in detection accuracy.
The present invention is made to solve the above-mentioned problems and has for its object to provide a biomolecule-immobilized plate having a highly smooth surface on which functional groups are produced, a biomolecule-immobilized plate on which biomolecules are immobilized via the functional groups, and methods for fabricating biomolecule-immobilized plates by which these plates can be fabricated easily and inexpensively.